Green fluorescent protein (GFP) is an intracellular reporter molecule widely utilized for assessment of gene transfer and expression. Enhanced variants have been cloned into various expression vectors suited for many different cell types. To study the effect of a gene of interest on cell cycle progression, it is desirable to measure GFP expression in combination with DNA content. This approach is difficult, as most suitable fluorescent DNA dyes are too large to pass through intact cell membranes, but permeabilization will allow GFP to leak out. The authors present a protocol with a cell preparation technique designed to maintain the delicate balance between retaining GFP fluorescence and obtaining adequate DNA histogram resolution. An Alternate Protocol describes a combined GFP fluorescence and cell cycle analysis using unpermeabilized cells stained with the vital dye Hoechst 33342.